How MS/MS spectra can be used for peptide and protein identification Peptide Sequencing How MS/MS spectra can be used for peptide and protein identification Mass spectrometry has been used a lot in biology since the late 1950’s. However it really came into play in the late 1980’s once methods were developed to allow the analysis of large intact (bigger than 1,000 Daltons) molecule. Two soft ionization techniques, Electrospray and Matrix Assisted Laser Desorption led to a huge jump in popularity as did the development of much more compact (bench top rather than whole laboratory) mass spectrometers.

What is Mass? Mass is given as m/z which is the mass of the ion divided by its charge Monoisotopic mass is the mass of an ion for a given empirical formula calculated using the exact mass of the most abundant isotope of each element (C=12.00000, H=1.007825 etc) Average mass is the mass of an ion for a given empirical formula calculated using the average exact mass for each element (C=12.01115, H=1.00797 etc) Nominal mass is the mass of an ion for a given empirical formula calculated using the integer mass of the most abundant isotope for each element (C=12, H=1 etc) Mass spectrometry is dependant on the ability to turn the analyte of interest into individual but intact, charged molecules in the gas phase. These are released into a low pressure area (a vacuum from 10-3 to 10-10 torr) where they can be manipulated by electrostatic and/or magnetic fields and separated. The force fields require the molecule to be charged, neutral molecules cannot be manipulated and are lost from the system.

Isotopes and Small Molecules Here the spectrum of a small molecule caffeine, shows basically one main peak and a few larger but much less intense peaks.

Isotopes and Peptides Isotope distributions in nature: Carbon C12 (98.9%), C13 (1.1%), C14 (small) Hydrogen H1 (99.98%), Deuterium (0.015%), Tritium (small) Oxygen O16 (99.8%), O17 (0.04%), O18 (0.2%) Sulphur S32 (95.0%), S33 (0.8), S34 (4.2%) These atoms are common in biological systems. Since the heavy isotopes are rare, this is not significant for most small molecules. Once one starts to look at biological molecules like peptides, the mass changes can be significant. For example, insulin has a mass of over 6,000 and thus a 1% shift by each of carbon, oxygen, hydrogen etc. can spread the mass from the lightest molecule (all C12, H1, O16 etc) to the heaviest (all C13, D2, O18 etc) over a range of 20-30 mass units. However some elements such as bromine have almost equally distributed isotopes (Br79 50.5% and Br 81 49.5%) which give rise to spectra with all peaks appearing as doublets. The effect of the isotope distribution on the shape of the spectrum (sometimes called the isotope envelope) becomes much more pronounced when analysing larger biomolecules. Here the various masses are shown for the peptide hormone glucagon.

Electrospray Ionisation and Charge Substance P 300 400 500 600 700 800 900 1000 1100 1200 1300 Da/e 100 % 674.7 666.1 600.4 462.8 685.7 693.6 1347.7 [M+2H]2 + [M+H] + Here is a spectrum of pure substance P, a peptide. Since a mass spectrometer always measures m/z the mass to charge ratio, two peaks are found. Here we see two main peaks, the doubly and singly charged ions.

Determining Charge State 520 521 522 523 524 525 526 527 528 529 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 524.3 525.3 526.2 Single Charge State Delta = 1.0 amu Delta = 1.0 amu In order to find out what you are looking at, i.e. is it singly, doubly etc charged, one looks at the details of the isotope distribution. Since isotopes are always one mass unit apart, if the peaks are one unit apart, the ion is singly charge since the mass difference (1 mass unit) divided by the charge (1+) is 1. Delta = 1.0 amu m/z

Determining Charge State 258 259 260 261 262 263 264 265 266 267 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 262.6 263.1 263.6 Double Charge State Delta = 0.5 amu Delta = 0.5 amu If the peaks are 0.5 mass units apart, they are doubly charged. Remember m/z,that the mass difference is 1 unit between isotopes and if the charge is 2 then the the mass change is 1/2 = 0.5 Delta = 0.5 amu m/z

How a Mass Spectrometer works....... An electric field accelerates the ions to a high speed. After this, they are directed into a magnetic field which applies a force to each ion perpendicular to the plane defined by the particles' direction of travel and the magnetic field lines. This force deflects the ions (makes them curve instead of traveling in a straight line) to varying degrees depending on their mass-to-charge ratio. Lighter ions get deflected more than the heavier ions. This is due to Newton's second law of motion. The acceleration of a particle is inversely proportional to its mass. Therefore, the magnetic field deflects the lighter ions more than it does the heavier ions. The detector measures the deflection of each resulting ion beam. From this measurement, the mass-to-charge ratios of all the ions produced in the source can be determined.

Single Stage Mass Spectrometry protein peptides peptides + + + + + + + + Mass Analysis Digestion Ionization The simplest type of mass spectrometer involves a single mass separation stage. The ions that are passed from the source into the mass analyzer give a simple read out of the intact molecular ions (assuming the ionization method is soft enough) MS m/z

Tandem Mass Spectrometry peptide fragments protein peptides ++ + + + ++ ++ + + + + + + + + ++ + Mass Analysis Digestion Ionization Isolation Fragmentation As interest grew in analyzing the structure of molecules, more complex mass spectrometers were developed with two mass separation stages. The first stage allows the selection of unique molecules by creating a single mass window to filter away other molecular species. The isolated molecule can then be broken into smaller components by a variety of techniques and the resultant fragment ions can be analysed in the second mass seperation stage. This is called tandem mass spectrometry or MS/MS since it originally was carried out using two mass spectrometers joined together in tandem. MS Isolation MS/MS m/z m/z m/z

Tandem Mass Spectrometry and Fragmentation Fragmentation of gas-phase ions is essential to tandem mass spectrometry and occurs between different stages of mass analysis. There are many methods used to fragment the ions and can result in different types of fragmentation and thus different information about the structure and composition of the molecule.  There are a number of different tandem MS experiments, which each have their own applications and offer their own information. An instrument equipped for tandem MS can still be used to run MS experiments. Tandem MS can be done in either time or space. Tandem MS in space involves the physical separation of the instrument components (QqQ or QTOF), tandem MS in time involves the use of an ion trap. Post-source fragmentation is most often what is being used in a tandem mass spectrometry experiment. Energy can also be added to the, usually already vibrationally excited, ions through post-source collisions with neutral atoms or molecules, the absorption of radiation, or the transfer or capture of an electron by a multiply charged ion. Collision-induced dissociation (CID), also called collisionally activated dissociation (CAD), involves the collision of an ion with a neutral atom or molecule in the gas phase and subsequent dissociation of the ion. In mass spectrometry, collision-induced dissociation (CID), referred to by some as collisionally activated dissociation (CAD), is a mechanism by which to fragment molecular ions in the gas phase. The molecular ions are usually accelerated by some electrical potential to high kinetic energy in the vacuum of a mass spectrometer and then allowed to collide with neutral gas molecules (often helium, nitrogen or argon). In the collision some of the kinetic energy is converted into internal energy which results in bond breakage and the fragmentation of the molecular ion into smaller fragments. These fragment ions can then be analyzed by a mass spectrometer. In peptide analysis, CID cleaves randomly along the peptide backbone producing b and y ions (see later section 6) and can cause loss of modifications (and amino acid side-chain fragmentation if very high energy is used). CID and the fragment ions produced by CID are used for several purposes. Partial or complete structural determination can be achieved. In some cases identity can be established based on previous knowledge without determining structure. Another use is in simply achieving more sensitive and specific detection. By looking for a unique fragment ion you can detect a given molecule in the presence of other molecules of the same nominal molecular mass, essentially reducing the background and increasing the limit of detection. Electron transfer dissociation (ETD) is a method to fragment ions in a mass spectrometer. Similar to electron capture dissociation, ETD induces fragmentation of cations (e.g. peptides or proteins) by transferring electrons to them. ETD does not use free electrons but employs radical anions (e.g. anthracene or azobenzene) for this purpose. Anthracene is vapourised and subjected to electron bombardment to generate anthracene carrying an extra electron. This charged molecular ion is introduced in the collision cell and transfers an electron to the peptide to be analysed. ETD cleaves randomly along the peptide backbone (so called c and z ions) while side chains and modifications such as phosphorylation and glycosylation are left intact.

Analyse Fragment Masses Tandem in Space Analyse Parent Masses MS Select Parent Fragment Analyse Fragment Masses MS/MS Two types of MS/MS experiments can be carried out depending on the instrument type being used. The first approach developed was tandem in space, in which the parent molecule of interest is fragmented in one part of the instrument before being moved to a second part for the analysis of the daughter (fragment) ions.

Tandem in Time Isolate Parent Ion Intact ions Intact ion This analysis/isolation/fragmentation can be carried out in an ion trap Fragment 426.7 The alternative to tandem in space, is the type of experiment that is carried out in an ion trap; tandem in time. Here the isolation of the parent molecule and the analysis of the daughter ions produced by fragmentation occur in the same part of the instrument. The two processes are merely separated by time, the parent isolation occurs first, then the fragmentation and finally the daughter analysis is carried out in the same part of the trap. Daughters

Tandem Mass Spectrometry (MSn ) 50 100 150 200 250 300 350 m/z % 284 282 142 107 71 249 214 177 253 288 MS full scan Fragments 50 100 150 200 250 m/z % MS2 Daughters of 284 50 100 150 200 250 m/z % MS3 Daughters of 232 of fragments 50 100 150 200 250 m/z % MS4 Daughters of 172 of fragments.... If a trap is being used, the cycle of –Isolation-Fragmentation-Detection can be run several times. A parent ion can be isolated and fragmented and the daugther ions analysed to generate a normal MS/MS spectrum. A daughter ion in this MS/MS spectrum can then be subsequently isolated, fragmented and the daughters of the daughter ion measured. This is termed MS/MS/MS or MS3. Experiments with a depth of MS7 have be carried out.

Peptide Fragmentation

Peptide charge in solution and gas phase Tryptic Peptide N-term C-term ETAGDPFFK 0--00-0000- at pH10 net charge -4 +-000-000+- at pH7 net charge -1 +00000000+0 at pH1.5 net charge +2 000000+00+0 gas phase net charge +2

Peptide Bond Formation on the Ribosome 3 N + C C O - R 1 2 O - H O 2 H O R 1 3 N + C C N C C O - 2 Amide Linkage O R 2 H 3 N + C C N C C O - 1

Calculating peptide masses H C OH O R1 R2 R3 +1 H+ +17 +1 Residue Mass 1 Residue Mass 3 Residue Mass 2 Residue Mass 4 Peptide mass = Sum of residue masses +1+1+17

Bond strengths in water and gas Bond Type Length (nm) Strength (kcal/mol) In vacuum In water Covalent ~0.15 90 Ionic ~0.25 80 3 Hydrogen ~0.30 4 1 Van der Waals ~0.35 0.1

Fragmentation Mode (MS/MS) Capillary HPLC Sample Ionisation Analyse Parent Masses (ii) MS/MS mode Select Parent Fragment Analyse Fragment Masses (i) MS mode

Peptide Fragmentation (MS/MS) Random fragmentation along the peptide backbone of ABCDE2+ +ABCDE+ N H 2 C O R 1 3 4 a b c z x y +A BCDE+ +AB CDE+ +ABC DE+ +ABCD E+ b-ions y-ions H

MS/MS Spectrum CID F L G K + b1 b2 b3 y3 y2 y1 b1 b2 b3 y1 y2 y3 L F K Relative Intensity m/z

Peptide Fragmentation by MS/MS 887.6 100 y10 90 986.6 80 y8 70 y12 b5 774.5 Relative Abundance 60 y12* 1186.7 494.3 y11 b6 50 1168.7 y13 1085.7 y7 607.4 1243.7 40 673.5 b4 b11 30 b8 b12 395.2 b5* b6* 1006.6 b7 1119.6 b13 20 476.3 779.5 589.3 772.5 885.6 a5 708.4 984.6 a12 1184.7 10 466.3 b10 400 500 600 700 800 900 1000 1100 1200 1300 1400 His-Gly-Thr-Val-Val-Leu-Thr-Ala-Leu-Gly-Gly-Ile-Leu-Lys b1 b4 b3 b5 b6 b7 b8 b9 b10 b2 b11 b12 b13 y13 y10 y11 y9 y8 y7 y6 y5 y4 y12 y3 y2 y1 m/z

MS/MS spectrum of m/z 593.8 Jr_74 # 888-890 RT: 22.70-22.74 AV: 2 NL: + c d Full ms2 593.8@35.00 [ 150.00-1200.00] 200 300 400 500 600 700 800 900 1000 1100 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 968.2 584.9 969.2 653.2 766.2 218.8 654.1 881.2 190.9 534.0 475.9 877.2 439.1 970.2 310.1 585.8 420.9 767.2 516.0 1039.2 730.3 950.2 220.0 617.1 305.9 403.1 882.2 1021.1 841.1 292.0 462.9 822.3 385.7 261.1 714.2 694.1 894.1 341.1 1042.2 859.0 748.1 635.1 552.1

Step 1: determine peptide mass (M+H)+ Jr_74 # 888-890 RT: 22.70-22.74 AV: 2 NL: 4.66E5 T: + c d Full ms2 593.8@35.00 [ 150.00-1200.00] 200 300 400 500 600 700 800 900 1000 1100 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 968.2 584.9 969.2 653.2 766.2 218.8 654.1 881.2 190.9 534.0 475.9 877.2 439.1 970.2 310.1 585.8 420.9 767.2 516.0 1039.2 730.3 950.2 220.0 617.1 305.9 403.1 882.2 1021.1 841.1 292.0 462.9 822.3 385.7 261.1 714.2 694.1 894.1 341.1 1042.2 859.0 748.1 635.1 552.1 [M+H]+ = 593.8 x 2 –1 = 1186.6

Step 2: Can we assign N-terminus aa? Jr_74 # 888-890 RT: 22.70-22.74 AV: 2 NL: 4.66E5 T: + c d Full ms2 593.8@35.00 [ 150.00-1200.00] 200 300 400 500 600 700 800 900 1000 1100 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 968.2 584.9 969.2 653.2 766.2 218.8 654.1 881.2 190.9 534.0 475.9 877.2 439.1 970.2 310.1 585.8 420.9 767.2 516.0 1039.2 730.3 950.2 220.0 617.1 305.9 403.1 882.2 1021.1 841.1 292.0 462.9 822.3 385.7 261.1 714.2 694.1 894.1 341.1 1042.2 859.0 748.1 635.1 552.1 a2/b2 combo present? a2/b2 combo Dm = 28 218.8 – 190.9 = 28 a2/b2

Step 2. What are the first 2 aa? [b2]+ = 219 Possible combinations: A + F M + S D + C Which one is correct? Can I see that aa as corresponding yn-1 ion? (A) 1186.6 – 71 = 1115.6 (F) 1186.6 – 147 = 1039.6 (M) 1186.6 – 131 = 1055.6 (S) 1186.6 – 87 = 1099.6 (D) 1186.6 – 115 = 1071.6 (C) 1186.6 – 103 = 1083.6

Step 2. Looking for y-1 ion in the spectrum Jr_74 # 888-890 RT: 22.70-22.74 AV: 2 NL: 4.66E5 T: + c d Full ms2 593.8@35.00 [ 150.00-1200.00] 968.2 700 750 800 850 900 950 1000 1050 1100 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance (A) 1186.6 – 71 = 1115.6 (F) 1186.6 – 147 = 1039.6 (M) 1186.6 – 131 = 1055.6 (S) 1186.6 – 87 = 1099.6 (D) 1186.6 – 115 = 1071.6 (C) 1186.6 – 103 = 1083.6 969.2 766.2 881.2 859.0 877.2 970.2 767.2 859.9 748.1 730.3 950.2 1039.2 749.1 768.3 822.3 841.1 882.2 951.3 994.0 1021.1 1042.2 694.1 714.2 731.1 804.2 894.1 943.0

Step 2. N-terminus assigned as FA… Jr_74 # 888-890 RT: 22.70-22.74 AV: 2 NL: 4.66E5 T: + c d Full ms2 593.8@35.00 [ 150.00-1200.00] 700 800 900 1000 1100 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 968.2 584.9 F FA 969.2 653.2 766.2 218.8 654.1 881.2 190.9 475.9 534.0 310.1 439.1 552.1 420.9 585.8 635.1 877.2 970.2 516.0 748.1 767.2 859.0 730.3 1039.2 220.0 305.9 950.2 403.1 617.1 841.1 882.2 1021.1 261.1 292.0 341.1 385.7 462.9 694.1 714.2 822.3 894.1 1042.2 200 300 400 500 600

Step 3. What is on C-terminus? Assuming tryptic peptide: (K) y1 = 128 + 18 + 1 = 147 (R) y1 = 156 + 18 + 1 = 175 High mass region: bn-1 ion (K) bn-1 = 1186.6 – 18 – 128 = 1040.6 (R) bn-1 = 1186.6 – 18 – 156 = 1012.6

Step 3. Looking for y1 ion… (K) y1 = 147 (R) y1 = 175 Not visible Jr_74 # 888-890 RT: 22.70-22.74 AV: 2 NL: 4.66E5 T: + c d Full ms2 593.8@35.00 [ 150.00-1200.00] 200 300 400 500 600 700 800 900 1000 1100 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 968.2 584.9 969.2 653.2 766.2 218.8 654.1 881.2 190.9 534.0 475.9 877.2 439.1 970.2 310.1 585.8 420.9 767.2 516.0 1039.2 730.3 950.2 220.0 617.1 305.9 403.1 882.2 1021.1 841.1 292.0 462.9 822.3 385.7 261.1 714.2 694.1 894.1 341.1 1042.2 859.0 748.1 635.1 552.1 (K) y1 = 147 (R) y1 = 175 Not visible

Step 3. C-terminus assigned… Jr_74 # 888-890 RT: 22.70-22.74 AV: 2 NL: 4.66E5 T: + c d Full ms2 593.8@35.00 [ 150.00-1200.00] 968.2 700 750 800 850 900 950 1000 1050 1100 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance bn-1 = 1040.6 bn-1 = 1012.6 969.2 K on C-terminus 766.2 881.2 859.0 877.2 970.2 767.2 859.9 748.1 730.3 950.2 1039.2 749.1 882.2 951.3 1021.1 768.3 822.3 841.1 994.0 1042.2 694.1 714.2 731.1 804.2 894.1 943.0

Step 4. Extending the y-ion series Jr_74 # 888-890 RT: 22.70-22.74 AV: 2 NL: 4.66E5 T: + c d Full ms2 593.8@35.00 [ 150.00-1200.00] 700 800 900 1000 1100 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 968.2 584.9 E X T X D S A F 969.2 653.2 D113 D71 D115 766.2 218.8 D87 D101 654.1 881.2 190.9 D129 D113 475.9 534.0 310.1 439.1 552.1 585.8 635.1 877.2 970.2 420.9 516.0 748.1 767.2 859.0 220.0 730.3 1039.2 305.9 403.1 617.1 950.2 841.1 882.2 1021.1 261.1 292.0 341.1 385.7 462.9 694.1 714.2 822.3 894.1 1042.2 200 300 400 500 600

Step 4. Finishing the y-ion series m/z 310.1 is made of (at least) 2 amino acids, one of them being R or K m/z 310.1 from y-series 310.1 = aa + K + 18 + 1 or 310.1 = aa + R + 18 + 1 aa = 163.1 aa = 146.1 Tyr = 163.1 Peptide ends with K

Step 4. Finishing the y-ion series Jr_74 # 888-890 RT: 22.70-22.74 AV: 2 NL: 4.66E5 T: + c d Full ms2 593.8@35.00 [ 150.00-1200.00] 700 800 900 1000 1100 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 968.2 584.9 K, Y E X T X D S A F 969.2 653.2 766.2 218.8 654.1 881.2 190.9 475.9 534.0 310.1 439.1 552.1 585.8 635.1 877.2 970.2 420.9 516.0 748.1 767.2 859.0 220.0 730.3 1039.2 305.9 617.1 950.2 403.1 841.1 882.2 1021.1 261.1 292.0 341.1 385.7 462.9 694.1 714.2 822.3 894.1 1042.2 200 300 400 500 600

Step 4. b-ion series K, Y E X T X D S A F F A S D X T X E Y K Jr_74 # 888-890 RT: 22.70-22.74 AV: 2 NL: 4.66E5 T: + c d Full ms2 593.8@35.00 [ 150.00-1200.00] 700 800 900 1000 1100 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 968.2 584.9 K, Y E X T X D S A F F A S D X T X E Y 969.2 K 653.2 766.2 218.8 654.1 881.2 190.9 475.9 534.0 310.1 439.1 552.1 585.8 635.1 877.2 970.2 420.9 516.0 748.1 767.2 859.0 1039.2 220.0 730.3 305.9 403.1 617.1 950.2 841.1 882.2 1021.1 261.1 292.0 341.1 385.7 462.9 694.1 714.2 822.3 894.1 1042.2 200 300 400 500 600

Step 5. Check sum of amino acid masses Jr_74 # 888-890 RT: 22.70-22.74 AV: 2 NL: 4.66E5 T: + c d Full ms2 593.8@35.00 [ 150.00-1200.00] 1186.6 700 800 900 1000 1100 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 968.2 584.9 K, Y E X T X D S A F F A S D X T X E Y 969.2 K 653.2 766.2 218.8 654.1 881.2 190.9 475.9 534.0 310.1 439.1 552.1 585.8 635.1 877.2 970.2 420.9 516.0 748.1 767.2 859.0 1039.2 220.0 730.3 305.9 403.1 617.1 950.2 841.1 882.2 1021.1 261.1 292.0 341.1 385.7 462.9 694.1 714.2 822.3 894.1 1042.2 200 300 400 500 600

Step 5. Features confirming the sequence Jr_74 # 888-890 RT: 22.70-22.74 AV: 2 NL: 4.66E5 T: + c d Full ms2 593.8@35.00 [ 150.00-1200.00] Loss of H2O from parent ion 700 800 900 1000 1100 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 968.2 584.9 K, Y E X T X D S A F F A S D X T X E Y 969.2 K 653.2 766.2 218.8 654.1 881.2 190.9 475.9 534.0 310.1 439.1 552.1 585.8 635.1 877.2 970.2 420.9 516.0 748.1 767.2 859.0 1039.2 220.0 730.3 305.9 403.1 617.1 950.2 841.1 882.2 1021.1 261.1 292.0 341.1 385.7 462.9 694.1 714.2 822.3 894.1 1042.2 200 300 400 500 600